Project Summary/Abstract Blood vessels play critical developmental roles in organogenesis related to cellular differentiation, tissue morphogenesis, and stem cell niches, in addition to delivering oxygen and metabolites to developing tissues. Dysregulation of vascular development is associated with a number of birth defects and is intimately linked to tumor progression and metastasis. In spite of these broad human health implications, the mechanisms that underlie vasculature?s instructive roles in essential biological processes are poorly understood and represent a major knowledge gap in the field. Our recent work in mice demonstrated that vascular-mesenchymal crosstalk is a vital process during fetal testis organogenesis. When the male sex determination pathway is triggered in the presumptive testis, endothelial cells migrate into the testis to form new vascular networks. Interestingly, vascular remodeling does not occur in the ovary at the same developmental stage. It has been proposed that Sertoli cells, the supporting cells of the testis, are the main drivers of testis organogenesis, but our recent work showed that vascular-mesenchymal interactions are a critical morphogenetic force in testis formation. Whether instructive roles for vasculature exist in other contexts in the fetal testis is unclear. Our preliminary data reveal a novel role for testicular vasculature in maintaining progenitors for Leydig cells (LCs), which are steroidogenic cells in the testis interstitial compartment required for the production of testosterone, a critical hormone for sexual differentiation and adult fertility. When we blocked vascular development of the early fetal testis, supernumerary LCs differentiated and fewer progenitor cells were maintained, strongly suggesting that blood vessels are a critical component of the LC niche. However, the molecular and cellular underpinnings of vasculature?s instructive capacity are unknown. Our long-term goals are to understand which cell types are essential for maintaining the LC niche and to uncover the molecular signals that regulate LC differentiation. We will test our central hypothesis that vascular-mesenchymal crosstalk is required for the establishment and maintenance of the LC niche through 2 specific aims: 1) to delineate the cell types that comprise the vascular niche required for the establishment and maintenance of LC progenitors; and 2) to elucidate the molecular mechanisms underlying endothelial-progenitor interactions within the LC niche, focusing on Notch signaling between endothelial cells and interstitial mesenchyme. To accomplish these aims, we will employ: in vivo genetic mouse models; ex vivo whole organ culture systems; in vitro primary cell co-culture techniques; and whole organ time-lapse live imaging. These approaches will allow us to uncover key molecular signals and cellular players that underlie vasculature?s vital developmental role in a tissue stem cell niche, which we anticipate will have direct relevance for the study of disorders of sexual development (DSDs) and other birth defects of the reproductive system, hormone-related male infertility, and vascularization-associated events in organogenesis and tumorigenesis.